Bacterial communities of Antarctic lichens explored by gDNA and cDNA 16S rRNA gene amplicon sequencing

Abstract Recently, lichens came once more into the scientific spotlight due to their unique relations with prokaryotes. Several temperate region lichen species have been thoroughly explored in this regard yet, the information on Antarctic lichens and their associated bacteriobiomes is somewhat lacking. In this paper, we assessed the phylogenetic structure of the whole and active fractions of bacterial communities housed by Antarctic lichens growing in different environmental conditions by targeted 16S rRNA gene amplicon sequencing. Bacterial communities associated with lichens procured from a nitrogen enriched site were very distinct from the communities isolated from lichens of a nitrogen depleted site. The former were characterized by substantial contributions of Bacteroidetes phylum members and the elusive Armatimonadetes. At the nutrient-poor site the lichen-associated bacteriobiome structure was unique for each lichen species, with chlorolichens being occupied largely by Proteobacteria. Lichen species with a pronounced discrepancy in diversity between the whole and active fractions of their bacterial communities had the widest ecological amplitude, hinting that the nonactive part of the community is a reservoir of latent stress coping mechanisms. This is the first investigation to make use of targeted metatranscriptomics to infer the bacterial biodiversity in Antarctic lichens.


Introduction
Lic hens, or r ather lic henized fungi (the fungus being the main morphology-and classification-determining component), are one of the most nonconspicuous, yet r elativ el y pr oductiv e elements of terrestrial habitats worldwide (Asplund and Wardle 2017 ). They ar e primaril y defined as a self-sustaining, m utualistic symbiosis between an Ascomycete fungus (mycobiont) and a photobiont, which can either be a green algae (chlorolichens), a c y anobacterium (c y anolic hens), or both (tripartite lic hens). These or ganisms, r epr esenting differ ent kingdoms, associate into c har acteristic and species-specific structures, called the vegetative thalli. Both components are highly codependent, as the mycobiont provides a microhabitat, as well as water and mineral salts, whereas the photobiont supplies photosynthetically fixed carbon in the form of sugars and sugar alcohols (Asc henbr enner et al. 2016, Honegger 1991. Lichenization seems to be a very successful surviv al str ategy among fungi, as 18% of the recognized species are capable of this kind of symbiosis, which can be traced back at least 415 million years, to the early Devonian era (Ha wks worth 2001 , Honegger et al. 2013 ). Lichens display an epiphytic or epilithic lifestyle, growing on the barks of trees, on bare rock, or compacted soil. They are estimated to cover up to 8% of the Earth's terrestrial landscape (Ahmadjian 1995 ).
The redefinition of what makes up a lichen has been established onl y r ecentl y, in lar ge part due to the emer gence of meta-"omic" tec hnologies (Cernav a et al. 2017, Grimm et al. 2021. By the updated definition, presented by Ha wks worth and Grube ( 2020 ) lic hens ar e "a self-sustaining ecosystem formed by the inter action of an exhabitant fungus and an extracellular arrangement of one or more photosynthetic partners and an indeterminate number of other microscopic organisms," amending the classic definition with the existence of a div erse, intr athallic consortium of micr oscopic or ganisms, consisting of nonphotosynthetic bacteria and archaea, accessory fungi and algae (Grimm et al. 2021 ). Sever al r esearc hers indicate that this m ultior ganismal consortium lik ely d wells within the lic hen thallus, surr ounded by extr acellular substances secreted by more than one component of this consortium. This complex biofilm layer composed of pol ysacc harides, such as glucans and mannans, can be considered an extracellular inter action matrix, pr oviding a medium, in whic h an exc hange of nutrients and signaling molecules takes place between different partners of the symbiosis (Tuovinen et al. 2019, Spribille et al. 2020. By employing culture-independent methods, a substantial part of this ne wl y discov er ed lic hen-associated comm unity was identified as heter otr ophic bacteria (P ankr atov et al. 2017 ). Intraand extrathallic activity of these bacteria may be of pivotal importance for lichen growth and survival. Metaproteomic and culturebased studies sho w ed lichen bacteriomes' potential for dinitrogen fixation and r oc k weathering-featur es involv ed in biogenic element acquisition, consequently making them available to lichen partners (Liba et al. 2006, Grube et al. 2015, Eymann et al. 2017. Lytic acti vities lik e c hitynol ysis, pr oteol ysis, and glucanol ysis also have been noticed, which presumably aid in recycling of old thalli parts (Sigurbjörnsdóttir et al. 2016 ). Lichen-associated bacteria are also responsible for vitamin and cofactor supply to the thallus, as well as furthering the growth and de v elopment of the lic hen thr ough phytohormone pr oduction Ber g 2009 , Noh et al. 2021 ). Resistance to many abiotic factors, such as low temper atur es, desiccation, and o xidati v e str ess (Eymann et al. 2017, Grimm et al. 2021 ) may also be mediated by lichenophilic bacteria. Additionall y, thr ough the pr oduction of secondary metabolites, some with antagonistic effects on other micr oor ganisms, the bacteriome poses as a major factor in the formation of the lichen species-specific microbiome (Grube et al. 2019 ).
Despite there being over 20 000 species described, lichens are often overshado w ed in their natural habitats b y the highl y div erse vascular plants (Cornelissen et al. 2007, Grimm et al. 2021. Howe v er, in high latitude regions, like Antarctica, the opposite is true (Armstr ong 2017 ). With ov er 400 species detected, lic hens v astl y outnumber resident plants in regard to biodiversity, since Antarctica houses only two species of flo w ering plants-Deschampsia antarctica and Colobanthus quitensis (Alberdi et al. 2002 ). Despite being very successful in extreme and cold habitats, lichen distribution in the Antarctic r egion, similarl y as in the remaining parts of the terrestrial biosphere, is governed by several external factors. Due to the lack of roots, an epidermis nor a cuticle, the lichens' metabolic activity is highly influenced by the local microclimate, geological substrate, as well as hydrological r egime, among others (Armstr ong 2015 ). Ho w e v er, the main factor which contributes to the formation of specific lichen communities in Antarctica, is the presence or the absence of a peculiar form of nitrogen enrichment (Bokhorst et al. 2019 ). This enrichment, experienced mainly at old and contemporary penguin nesting sites, is caused by the ammonification of uric acid from bird guano. Released ammonia v a pors cr eate a phenomenon known as an "ammonia shadow," which fertilizes r oc ks and gr ound surfaces near large penguin colonies (Tatur et al. 1997, Grzesiak et al. 2020. In suc h conditions, ornithocopr ophilous comm unities that ar e highl y nitr ophilic and cannot be found else wher e in the otherwise barren Antarctic landscape, develop. Nitrophobic communities also occur in the region, albeit can be found growing exclusiv el y wher e nutrient inflow is minor and rather indirect, therefor e, high nitr ogen concentr ations can be avoided, primaril y on r oc ky substr ates inland, at higher altitudes. Both of these comm unities ar e composed of, to a certain extent, endemic, specialist lichen species (obligatory nitrophiles or nitrophobes), while also containing nitr ogen-toler ant gener alist lic hen species, whic h gr ow r egardless of nitr ogen compound concentr ations (Olec h 2004, Johansson et al. 2011.
With the latest scientific liter atur e being incr easingl y amended with reports on lichen-associated microbiomes, the issue of how the bacterial communities' phylogenetic structure changes depending on nitrogen content preference of its host lichen has ne v er been in vestigated. T hus , with the use of up-to-date, highthr oughput tec hnologies, we assessed the phylogenetic structur e of the whole and active fraction of bacterial communities of Antarctic lic hens gr owing in differ ent envir onmental conditions. Our hypothesis states that nitrogen compound concentrations in the lichens preferred habitat exerts a greater influence on its associated bacteriobiome than the specific features of the host. We also hypothesize that lichens able to host a greater diversity of bacteria also have a wider niche/habitat range.
In order to pr ov e this, we carried out a targeted 16S rRNA gene amplicon sequencing based on genomic DN A (gDN A), as w ell as complimentary DN A (cDN A). gDN A w as consider ed as r epr esen-tative for the whole present bacterial community, whereas cDNA r epr esented the metabolicall y activ e part of this comm unity on the premise that only active bacterial cells harbored intact ribosomal RNA molecules-a substrate in the applied metatranscriptomic pr ocedur e. Nucleic acids wer e extr acted fr om bacterial comm unities r esiding in nitr ophilous, nitr ogen-toler ant, and nitr ogen-sensitiv e lic hen species, fr om two distinct habitats at King George Island, maritime Antarctica: the nitrogen abundant ar ea surr ounding the P oint T homas penguin rookery and the nitr ogen-depleted ar ea near J ar dine P eak. Using these complementary types of data (cDN A and gDN A) allo ws to confer a more holistic picture of the impact that the polar environment poses on the bacterial community residing within lichens, as well as the micr obe-host inter actions taking place in one of the world's harshest, yet most intriguing ecosystem.

Lichen sampling scheme
Samples were obtained during the 43rd expedition to the Polish Antar ctic Station "Ar cto wski," fr om ice-fr ee ar eas at the western shore of Admiralty Bay (King George Island, Antarctica), as well as the barr en terr ains that border the southern shor e of the Ezcurr a Inlet (Table 1 , Fig. 1 A). Lichen specimens were collected from areas with varying nutrient inputs (Olech 2004 ). Sampling was done within the span of 8 h in March of 2019. Samples from the nitr ophilous (ornitocopr ophilous) lic hen comm unities included the species: Ramalina terebrata Hook f. and Taylor ( Fig. 1 B), Gondwania regalis ( Caloplaca regalis ) (Vain.) Søchting, Frödén and Arup ( Fig. 1 C), and Turgidosculum complicatulum ( Mastodia tesselata ) (Nyl.) J. Kohlm. and E. Kohlm. (Fig. 1 D) pr ocur ed fr om the nutrient-ric h site of the Point Thomas penguin r ookery. Samples fr om the nitrophobic lic hen comm unities included the species: Leptogium puberulum Hue ( Fig. 1 F), Himantormia lugubris (Hue) I. M. Lamb ( Fig. 1 G), Usnea aur antiaco-atr a (J acq.) ( Fig. 1 H) and w ere collected at the nutrientlacking site near the J ar dine P eak (Southern shore of the Ezcurra Inlet). Thalli samples of the nitr ogen-toler ant lic hen species Usnea antarctica Du Rietz ( Fig. 1 E) were obtained from both sites . T hree specimens of a particular species were collected per site with the use of sterile tweezers and scissors . T halli samples were immediatel y submer ged into 30 ml of StayRNA™ solution (A&A Biotechnology) in 50 ml conical tubes and stored overnight in 4 • C, as suggested in the user's manual. On the following day, the samples where rid of the remaining solution and stored in −20 • C until further analysis. Taxonomic identification of the lichen specimens was carried out by Maria A. Olech.

Nucleic acid isolation
DN A and RN A w er e coextr acted fr om the StayRNA™ pr eserv ed lichen thalli samples. In brief, 0.2 g of the sampled thallus was ground with a sterile pestle and mortar, with the addition of liquid nitrogen and sterile sharp, garnet sand (Lysing Matrix A, MP Biomedicals). The ground sample was divided into roughly equal aliquots for DNA and RNA isolations.
RN A isolation w as carried out under a UV cabinet. All surfaces and equipment were cleaned with the labZap™ solution (A&A Biotec hnology). Appr oximatel y, 0.1 g of the ground sample was mixed with 1 ml of TRI REAGENT © (Molecular Research Center, Inc.) and incubated for 5 min/RT, after which 0.2 ml of chloroform was added and incubated for 3 min/RT. The samples were centrifuged at 12 000 × g /15 min/4 • C and the RNA-containing aqueous phase tr ansferr ed into a ne w micr ocentrifuge tube . T his Table 1. Sampling sites' c har acteristics. GR-Gondw ania regalis ; RT-Ramalina terebrata ; TC-Turgidosculum complicatulum ; UP-Usnea antar ctica , penguin rook ery; HL-Himantormia lugubris ; UA-Usnea aur antiaco-atr a ; LJ-Leptogium puberulum ; and UJ-Usnea antarctica , Jardine Peak.

Sample
Site  step was carried out twice. Next, 3 vol of 96% EtOH and 1/10th vol of 3 M sodium acetate (pH 5.2) solution were added, and the samples were left overnight in −80 • C. The following day, the samples w ere v ortexed and left to sit for 10 min/RT, after which they were centrifuged at 7500 × g /5 min/4 • C. The RN A w as visible as a small white pellet. The pellet had been washed thrice with 75% EtOH and centrifuged at 7500 × g /5 min/4 • C. The samples were air-dried in RT. The pellet was resuspended in 50 μl DEPC-treated, nuclease-free water (A&A Biotechnology) and incubated at 4 • C to solubilize the RNA. After vortexing the samples, the concentration and purity were checked on a NanoPhotometer ® NP80 (Implen). The quality of the isolated RN A w as further impr ov ed by the use of the Clean-up RNA Concentrator kit (A&A Biotechnology), which included a DNA removal step with the use of a DNase, and c hec ked once a gain on a NanoPhotometer ® NP80 (Implen).
To ensure that the RN A w as completely de pri ved of DNA, a PCR reaction was set up, with the RNA samples serving as a template and Esc heric hia coli dh5 α gDNA serving as a positiv e contr ol. PCR reaction had been conducted using the high specificity ready-touse mix PCR Mix Plus (A&A Biotechnology) in a final volume of 25 μl per reaction, according to the user's manual. The primers used were gene-specific primers: 16S_V3-F and 16S_V4-R, which cover positions 341-357F and 785-805R, r espectiv el y, according to E. coli 16S rRNA gene r efer ence sequence (Klindworth et al. 2013 ). PCR pr oduct pr esence was c hec ked on an a gar ose gel electr ophor esis. DNase-treated and -cleaned RN A samples w er e stor ed until further analysis in −80 • C. The reverse transcription was carried out with the use of QuantiNova Reverse Transcription Kit (Qiagen) containing random hexamer primers according to the manufacturer's instructions . T he obtained 24 cDN A samples w er e stor ed in −20 • C until further analysis. Total DN A w as isolated using the CTAB method, accor ding to the protocol by Wilson ( 2001 ) featured in Current protocols in Molecular Biolog y , after whic h a concentr ation and purity c hec k was conducted on a NanoPhotometer ® NP80 (Implen). The samples were cleaned up with a Clean-up Concentrator kit (A&A Biotechnology), according to the user manual and followed-up with another concentration and purity check. The obtained 24 gDNA samples were stored in −20 • C until further analysis.

16S rRNA gene targeted amplicon sequencing of cDNA and gDNA
For the Illumina 16S rRNA-targeted amplicon sequencing, a PCR reaction had been conducted in triplicate using the WALK pol ymer ase ( Pwo pol ymer ase with pr oof-r eading activity) (A&A Bioetchnology), in a final volume of 25 μl per reaction, according to the user's manual. The primers used were gene-specific primers: 16S_V3-F and 16S_V4-R, whic h cov er positions 341-357F and 785-805R, r espectiv el y, according to E. coli 16S rRNA gene r efer ence sequence (Klindworth et al. 2013 ). The primers contained the Illumina Nextera XT (Illumina, San Diego, USA) overhang adapter nucleotide sequences, both added to the primer pair sequences for compatibility with Illumina index and sequencing adapters. The amplification conditions for both sets of primers were as follows: initial denaturation at 95 • C/3 min, followed by 30 cycles of denaturation at 95 • C/30 s, annealing at 58 • C/30 s, and elongation 72 • C/30 s, with the final elongation at 72 • C/5 min. The obtained pr oducts wer e pooled in equimolar ratio within a particular lichen species (and site for generalist lichens-see Table 1 ) and indexed using the aforementioned Nextera XT barcodes (Illumina). Amplicon libraries were pooled and sequenced on the Illumina MiSeq instrument (Illumina) at the DNA Sequencing and Synthesis Fa-cility (Institute of Biochemistry and Biophysics , P olish Academy of Sciences), with the sequencing conducted in paired-end mode (2 × 300 bp) with the use of a v3 (600 cycles) chemistry cartridge, which allo w ed gener ation of long pair ed r eads full y cov ering 16S V3-V4 amplicons. A total of 16 amplicon sequence sets were obtained, eight based on gDNA and eight based on cDNA.

Da ta anal ysis
Raw sequencing data were cleaned, aligned, and classified automatically by the EzBioCloud platform using the PKSSU4.0 database (Yoon et al. 2017 ). Chimeric, low quality, and nontarget (c hlor oplast, mitoc hondrial, and arc haeal) amplicons wer e automatically discarded. The operational taxonomic unit (OTU) was defined as a group of sequences that exhibit greater than 97% similarity to each other. Illumina reads were deposited in the NCBI Sequence Read Arc hiv e (SRA) as BioProject PRJNA873246. All results were compiled using Excel 2016 (MS Office) for Windows. Corr elations between famil y-r ank sequence abundances deriv ed from gDN A and cDN A data w ere calculated using Pearson's correlation coefficient. Principal component analysis (PCA) of lichenassociated bacterial communities based on famil y-r ank gr oup abundances was performed using the singular value decomposition method. Data visualization and statistical analysis have been performed using the R software (R v.4.0.2) and the following packa ges: ggplot2, pheatma p, ggv enn, fmsb, Hmisc, ggpubr, corr plot, and autoplot (R Core Team 2013 ).

Results
Tar get r eads obtained in the gDNA anal ysis r anged between 7506 and 79 834, while those obtained in the cDNA analysis between 3750 and 40 484. Bacterial OTU numbers based on gDNA analysis r anged fr om 418 to 1817 (av. 895.4, sd. 500.6), based on cDNA the OTU numbers were in the range of 137-881 (av. 433.1, sd. 246.2). Lo w est discrepancies betw een gDN A and cDN A deri ved OTU n umbers were noted for R . terebrata and G . regalis (84 and 95, respectiv el y), while the highest in T . complicatulum (1164 OTUs) and L . puberulum (1109 OTUs) (Fig. 2 A).
T he PC A cluster ed the differ ent bacterial comm unities according to r elativ e abundances of famil y-r ank sequences (Fig. 3 B). Ramalina terebrata community sho w ed the greatest dissimilarity to the rest of the lichen-associated community with the Hymenobacteraceae r elativ e abundance being the most significant determining factor. The L . puberulum community was also noticeably distant, with the Nostocaceae r elativ e abundance significantl y influencing the clustering outcome. Acidobacteriaceae r elativ e abundance defined a cluster consisting of U . aur antiaco-atr a community (gDNA and cDNA results). The relative abundance of Acetobacteraceae was significant for the H . lugubris gDNA community placement, wher eas the r elativ e abundance of Alcaligenaceae and Xanthomonadaceae defined the U . antarctica community (gDNA and cDN A) at the P enguin rookery, as w ell as the cDN A-derived H . lugubris comm unity. A some what coher ent gr oup w as formed b y the Penguin rookery lichen-associated communities of G . regalis and T . complicatulum .
Similarities betw een DN A templates based on shared OTU numbers sho w ed se v er al phenomena (Fig. 4 A). Bacterial comm unities of the nitrophilic species G . regalis , R . terebrata , and T . complicatulum sho w ed m uc h similarity betw een DN A templates (635, 469, and 609 of shared O TUs , r espectiv el y), while the comm unities associated with the nitrophobic species H . lugubris , U . aurantiacoatra , and L . puberulum sho w ed r elativ el y low similarity between templates (143, 134, and 408 shared O TUs between templates , r espectiv el y). Ther e wer e site-specific differ ences in shar ed OTU numbers between templates for the gener alist lic hen comm uni-  puberulum , U_P-U . antarctica at penguin rookery, U_J-U . antarctica at J ar dine P eak, penguin r ookery-cor e OTUs of penguin r ookery lic hen communities, and J ar dine P eak-core OTUs of J ar dine P eak lic hen comm unities.
ties . T he U . antarctica community at the Penguin rookery shared 309 OTUs between the gDN A and cDN A template whereas the J ar dine P eak U . antarctica comm unity shar ed onl y 103 O TUs . T he number of OTUs shared between the four lichen species growing at the penguin rookery (gDN A-169; cDN A-102) w ere substantially higher than those shared by the lichen species growing in the J ar dine P eak area (gDN A-67; cDN A-11) (Fig. 4 B). Based on the gDNA analysis, U . antarctica bacterial communities shared 313 OTUs between sites, yet only 30 based on cDNA sequences. Only 27 OTUs were shared between the core communities of the penguin rookery and J ar dine P eak area for the gDN A template and one for the cDNA template (Fig. 4 C).

Discussion
Despite the extensive knowledge about the input of lichens into the Earth's ecosystem, there is still much to learn about these pioneering metaor ganisms, especiall y r egarding the div ersity of the taxa partaking in this multispecies symbiosis in Antarctica (Grimm et al. 2021 ). In this study, we showed how amending metagenomic information with metatranscriptomic data allows for a more detailed description of the heter otr ophic bacterial component that activ el y contributes to the lichen symbiosis . T his additional information is especially important when comparing lichen bacteriomes in different trophic surroundings, as most profound changes in diversity can be seen through the activity of specialized phylogenetic and/or functional fractions, something that can only be studied through transcriptomic analyses (Cernava et al. 2019 ).
T he P oint T homas penguin rookery is considered an Antarctic Specially Protected Area due to its unique biodiversity (Chwedorzewska and Korczak 2010 ). Organisms found at this site either thrive on, or tolerate the high concentrations of ammonia fumes that come off the decaying guano, making it a major environmental factor exerting pressure on this biocenosis (which is also influenced by marine factors, as it is located at the shore of Admiralty Bay) (Olech 2004, Myrcha et al. 2013, Grzesiak et al. 2020. Bacterial communities of lichens procured from the penguin rookery displayed a high degree of similarity (as shown in the PCA outcome), with the exception of the R . terebrata communities. Ne v ertheless, the amount of OTUs shared between all four communities was relatively high, particularly between their active fractions . T his suggests that a consortium of bacteria adapted to the pr e v alent conditions has been established ther e. A vital part of this group of bacteria belonged to the Bacteroidetes phylum, whic h hav e been found to dominate, or massiv el y contribute, to the bacterial communities of the Point Thomas penguin rookery lichens . T his phylum's occurrence has also been noted for the strictly marine lichens of the Brittany coast (West et al. 2018 ). Authors of this pa per discov er ed the highest Bacteroides abundance in marine lichens, hinting that seawater influences may be the cause of this phenomenon. Ho w e v er, other lic hens inv estigated by West et al. ( 2018 ) (inland c y anolichens and maritime, nitrophilous lichens of the genus Xanthoria ), were also considerably rich in Bacter oidetes. Giv en our findings, a more plausible explanation can be given, that the Bacteroides bacteria tend to occupy lichens experiencing a r elativ el y high abundance of nitrogen, be it of endogenic (c y anobiont N 2 fixation) or exogenic natur e. Additionall y, cDNA data r e v ealed that Bacter oidetes wer e tr anscriptomicall y activ e onl y in the penguin r ookery lic hens and in the examined c y anolichen L . puberulum . The findings regarding the U . antarctica bacterial community further corroborate this thesis, with specimens growing at the rookery housing a substantial amount of Bacteroidetes members, while specimens of the J ar dine P eak area carrying only minuscule amounts . T he extent of Bacteroidetes dominance, seen here in R . terebrata , has also been observed in the lichen Umbilicaria decussata (a nitrogen-tolerant species) procur ed fr om the shor e of Lützow-Holm Bay, coastal Queen Maud Land, continental Antarctica, wher e se v er al lar ge penguin and petr el br eeding colonies ar e located (He et al. 2022 ). The majority of the R . terebrata bacteriome sequences were assigned to the famil y Hymenobacteraceae , whic h wer e also pr esent in the thr ee other penguin rookery lichen species. Information on the involvement of Hymenobacteraceae members in the lichen symbiosis is limited, ho w e v er, a genome study on a fe w lic hen-associated Hymenobacter spp. isolates r e v ealed their potential resistance to UV radiation, as well as the ability to decompose complex pol ysacc harides , Ghimire et al. 2020. Ramalina terebrata grows on vertical or greatly inclined rock walls, which are periodically under intense sunlight irradiation (Olech 2004 ). The species is also known for producing usnic acid and ramalin as UV protectant and antio xidant, respecti vely (Quilhot et al. 1996, Paudel et al. 2011 ). T hese , and a few other compounds produced by R . terebrata were confirmed as having an antimicrobial effect (Paudel et al. 2010 ). Consequently, the combination of these abiotic and biotic influences may have led to the de v elopment of a bacterial community, i.e. low in diversity, yet containing many m ultir esistant members, making them almost the exclusive inhabitants of the R . terebrata thalli. Inter estingl y, U . decussata is also known as a gyrophoric acid producer, which displays potent antimicr obial activity (Olec h 2004, Rank o vi ć et al. 2008 ). T he abundance of Hymenobacteraceae did not correlate with the abundance of any other bacterial family that was considerably contributing to the Antarctic lichens' communities in this study, yet their abundance based on gDNA positiv el y corr elated with that based on cDN A data. This sho ws their potential to form monoculture-like assemblages in the lichen thalli, and that members of this family ar e activ e in lic hen-associated comm unities whene v er they ar e pr esent. Besides Bacter oidetes, the two other nitr ophilic lic hen species: T . complicatulum and G . regalis also harbored considerable amounts of bacteria identified as Armatimonadetes by the database used. Available information points to w ar d Armatimonadetes pr efer ence for nitr ophilic, sea coast-dwelling c hlor olic hens (West et al. 2018, He et al. 2022. Members of this group are rarely cultivated, so the knowledge on their environmental role and featur es ar e scar ce, ho w e v er, fairl y r ecentl y, a ne w member of this taxon was isolated from Antarctic soil-Abditibacterium utsteinense (Tahon et al. 2018 ). Coincidentally, one of the most abundant OTUs shared among the communities associated with the lichens of the P oint T homas penguin rookery examined in this study was identified as the genus Abditibacterium (data not shown). Physiological and genomic investigations revealed that A . utsteinense is welladapted to coping with environmental stresses present in Antarctica: o xidati ve and radiation stress, temperature fluctuations but also to toxic compounds (Tahon et al. 2018 ). The strain had a very narr ow pH gr owth r ange, hinting that this bacterium is adapted to a stable environment, not unlike a lichen thallus, for which the pH homeostasis is crucial for surviv al. Furthermor e, it contained the genetic basis for a versatile nitrogen metabolism, including ammonia transporters, as well as the ability to sequester ammonia by -glutamine synthesis, which is a way to reduce NH 4 + concentration, thus avoiding its toxic effects-a major issue for organisms experiencing the aforementioned ammonia shadow effect (Dahlman et al. 2003, Crittenden et al. 2015. The J ar dine P eak ar ea constitutes a highland plateau wher e on r oc ky substr ates, suc h as exposed r oc k faces , ridges , and stony slopes, nitr ophobic lic hen comm unities de v elop (Olec h 2004 ). Lic hen-associated micr obiomes sampled ther e wer e dominated b y Proteobacteria, ho w ever, the communities w ere mostly host species-specific, as indicated by the low numbers of shared O TU's , especially those based on cDNA data. The most distinctive bacterial community among the J ar dine P eak c hlor olic hens was found residing in U . aur antiaco-atr a . It was composed mostly of Acidobacteria. T his feature , to a greater or lesser extent, was also observed for the lichen species Cladonia borealis and Oc hrolec hia parallela , pr ocur ed fr om nitr ogen-deficient sites at King Geor ge Island (Antarctica) (Park et al. 2016 ). Additionally to the preference for nutrientdepleted habitats, these lichens have the ability to individually produce a variety of complex, bioactive, and acidic compounds (Olec h 2004 ). Consequentl y, the combination of low intrathallic pH, pr efer ence for oligotr ophy and inhibitory compound presence likely led to the establishment of an Acidobacteria-dominated community, due to the specific niche requirements of these bacteria (Kielak et al. 2016 ). Most of the Acidobacteria sequences from U . aur antiaco-atr a were identified as belonging to the Acidobacteriaceae family. The abundance of this family did not correlate with the abundance of any other family, yet as with the Hymenobacteraceae , their abundance by gDNA-based analysis did correlate with cDNA-based abundance, hinting their presence is not influenced by interactions with other microbes, but by abiotic-and lichenbased factors, with pH likely serving as the most critical one. Some culture-based studies indicate that members of this family can gr ow on lic hen-specific pol ymers like lic henan, as well as c hitin and cellulose (Belova et al. 2018 ). Himantormia lugubris displays a curious case, where the structure of the active part of the bacterial community is distinctly different than that of the whole community. The latter was especially rich in Acetobacteraceae (Alpha pr oteobacteria), while the activ e part of this community was defined by the abundances of Alcaligenaceae (Beta pr oteobacteria) and Xanthomonadaceae (Gamma pr oteobacteria). Suc h discr epancies in the comm unity structur e, wher e a biodiv erse r eservoir of dormant bacteria was created, indicates that H . lugubris undergoes seasonal changes within intrathallic conditions (Cruaud et al. 2020 ). What is known of H . lugubris ecophysiology is that it has gr eat toler ance for desiccation, as well as being cov er ed with snow for extended periods of time (Choi et al. 2015 ). It has also been concluded that the production of inhibitory phenolic compounds ceases in H. lugubris during sunlight exposure (Mateos et al. 1991 ). The associated bacterial community may, ther efor e, r espond to these changes by shifting the abundance of its active participants . Furthermore , the active part of the H . lugubris community bears a very close resemblance to the U . antarctica community of J ar dine P eak (whole and active part). The tw o families whose abundance defines these communities displayed a positiv e corr elation with eac h other, suggesting a symbiotic relation. Ho w e v er, onl y Xanthomonadaceae displayed a positive correlation between their gDNA/cDNA abundances, meaning that Alcaligenaceae are more dependent on their presence than vice versa . The abundance of Alcaligenaceae was noticeably higher in Jardine Peak specimens of U. antarctica and H. lugubris , suggesting these bacteria ar e adv anta geous in oligotr ophic conditions. Some members of this family are endosymbiotic and nitrogen fixing (Taulé et al. 2012 ). Curious in this scenario is the positive correlation with the Xanthomonadaceae , whic h wer e r ecognized as major anta gonists in lichen-associated communities, presumably defending the resour ces provided b y Alcaligenaceae . Another interesting matter was the abundance of the family Acetobacteraceae in both Usnea species and H . lugubris . Members of this family wer e activ e in U . antarctica at the penguin r ook ery, hinting the y may help the lichen cope with excess nitr ogen. Note worthy is the fact, that ammonia v a pors cause a substantial alkalization of the surr ounding ar ea, consequentl y c hallenging the pH homeostasis of the non-nitrophilic lichens (Sutton et al. 2020, do Vale Lopes et al. 2021. Usnea antarctica may, ther efor e, be this toler ant of suc h high nitrogen concentrations due to the acidifying action of the lichen-associated Acetobacteraceae (Kersters et al. 2006 ). In the nitrophobe H . lugubris this family was not considerably active at the time of sampling. Ho w e v er, if the melting snow accumulates alkaline substances (mainly ammonia ions) and introduces them in early summer into the thallus, the properties of the Acetobacteraceae might pr ov e v ery beneficial for the lichen (Crittenden 1998 ).
Usnea antarctica presents a case of a lic hen, i.e. toler ant of high nitr ogen concentr ations in its immediate envir onment (Olec h 2004 ). As seen in the PCA gr a ph, the bacterial comm unities of U. antarctica did differ between the two sites. While the amount of OTUs for both these communities was similar, the number of shared OTUs between them was less than half, with concomitant, substantial differences at the phylum level. The active part of the J ar dine P eak community was less diverse than that of the penguin rookery. In the lichen Umbillicaria rhizinata the opposite was observed, with the coastal (and nitrogen fertilized) specimens hosting a less diverse community than the inland ones (He et al. 2022 ). Ho w e v er, similarl y to our U . antarctica samples, the coastal/nitr ogen enric hed specimens harbor ed taxa affiliated with nitr ophilic lic hens (Armatimonadetes, Bacter oidetes), while the inland lic hens wer e dominated by Proteobacteria (He et al. 2022 ). T hus , shifting of the bacterial comm unity structur e in nitr ogen-toler ant lic hens (or an y wide amplitude lic hens) may be the basis for their efficient adaptability to changing environmental conditions . Furthermore , the findings of Cerna va et al. ( 2015 ) indicate that the diversity of lichen microbiota strongly contributes to the adaptability and flexibility of the host-lichen, thus, the greater the resident bacterial diversity, the wider the ecological amplitude of the lichen species. According to our data, the T . complicatulum bacterial community displayed the highest number of OTUs for the whole community (gDNA data). This lichen is known to have a bipolar distribution whilst being able to thrive in high nitrogen and high salinity habitats (Olech 2004 ). The L . puberulum community was also very diverse, and despite the lichen being endemic to Antarctica, it is v ery widespr ead in the region and can be found in a range of habitats, in both inland and coastal localities , pro vided there is an abundance of fresh water (Olech 2004 ). The third most diverse bacterial community, ho w e v er, was found in G . regalis -an ornitocoprophile, endemic to West Antarctica, restricted to sites with considerable ammonia exposure (Olech 2004 ). T herefore , the diversity of the whole community alone might be species-specific and not dictate the ecological amplitude of the host lichen. Ho w ever, the discrepanc y between the diversity of the whole and active community can suggest the scope of the ada ptiv e potential of the bacteriocenosis, as was indicated here for the nitrogen tolerant U . antarctica , which occupies a variety of Southern Hemisphere habitats and is not exclusive to Antarctica (Olech 2004 ).

Conclusions
Lastly, the bacterial communities of the penguin rookery lichens wer e distinct fr om the comm unities of the J ar dine P eak area lichens due to the small number of shared O TUs . En vironmental conditions present at the penguin rookery ( super omnia ammonia v a pors) lar gel y sha ped the lic hen-associated micr obiome, with some lichen species-specific traits, such as antimetabolite production, also influencing its structure. At the nutrient-deficient site of the J ar dine P eak the lichen-dw elling microbiocenoses w ere distinct for each lichen species . T he bacterial community of U . antarctica (found at both sites) displayed a high degree of flexibility. At the penguin rookery it resembled the communities found in nitr ophilous lic hens, while at the J ar dine P eak ar ea it harbor ed some bacterial groups found in nitrophobic lichens . T his trait ma y, ther efor e, be a k e y component in the adaptability of Usnea and other lichen species. Given that all of the sampled lichen species had an active and a dormant fraction of its resident bacterial community, it can be concluded that the latter r epr esents a r eservoir of latent, beneficial traits that once activated can widen the ecological amplitude of the lichen holobiont, thus expanding its habitat r ange, e v en to a worldwide extent.

Ac kno wledgments
Samples and data were obtained due to the scientific facility of the Polish Antarctic Station ARCTOWSKI.

Funding
This r esearc h was funded by the National Science Center, Poland (grant number 2017/25/B/NZ8/01915).